Peptide and osteogenetic accelerator

ABSTRACT

A peptide has any one of the sequences SEQ ID NO.1 to SEQ ID NO.8, or has a sequence derived from any one of the sequences SEQ ID NO.1 to SEQ ID NO.8 by substitution, deletion or addition of one or several amino acids therein and having an osteogenetic activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to Japanese application No. HEI10(1998)-322075 filed on Nov. 12, 1998, whose priority is claimed under35 USC §119, the disclosure of which is incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel peptide having osteogeneticactivity and an osteogenetic accelerator containing the same as anactive ingredient.

The peptide of the present invention, which has the osteogeneticactivity, is useful for treatment of fractures, as a filler in deficientsites of bone, for inhibition of decrease in bone substance related toosteoporosis and periodontic diseases, for prevention of fracturesassociated with osteoporosis and rheumatoid arthritis and the like.

2. Description of Related Art

Bone morphogenetic protein (BMP) is a member of transforming growthfactor (TGF) β family (Wozney, J. M. et al, Science, 242, 1528 (1988)),and its active form exists as a homodimer having a molecular weight ofabout 18kD. BMP has the function of acting on undifferentiatedmesenchymal cells, inducing differentiation to chondroblasts andosteoblasts and effecting chondrogenesis and osteogenesis (Wang, E. A.et al. Proc. Natl. Acad. Sci. USA, 87, 2220 (1990)).

For this reason, BMP is expected to be effective in treatment offractures, inhibition of decrease in the bone substance related toosteoporosis and periodontic diseases, in prevention of fracturesassociated with osteoporosis and rheumatoid arthritis and the like (forexample, see Japanese Unexamined Patent Publications Nos.

HEI 6(1994)-298800 and HEI 10(1998)-70989).

Also, there are known a number of inventions relating to implants andcompositions in which BMP is combined with a variety of matrices (forexample, see Japanese Unexamined Patent Publications Nos. HEI6(1994)-296677, HEI7(1995)-246235, HEI 7(1995)-116240, HEI 7(1995)-88174and HEI 10(1998)-151188).

However, the above-described BMP, when it is administered in vivo,disappears from blood within a few minutes and loses its effect. Ifadministered in a large amount for compensating that, BMP might possiblycause various adverse effects, including toxic effects on livers andkidneys. Further, BMP has an immunogenicity because of its largemolecular weight, and might possibly cause anaphylactic shock whenadministered repeatedly. furthermore, where BMP is impregnated inmatrices of decalcificated bone or collagen for use, osteogeneticactivity is expressed, but there may be another problem of antigenicityor infection attributed to the matrices.

SUMMARY OF THE INVENTION

Under these circumstances, an object of the present invention is toprovide a peptide having an osteogenetic activity in which peptide theabove-mentioned problems are alleviated, and an osteogenetic acceleratorcontaining the peptide.

According to the present invention, the above-mentioned object isachieved by a peptide having any one of the sequences SEQ ID NO.1 to SEQID NO.8.

Also the object of the present invention is achieved by an osteogeneticaccelerator containing the above-mentioned peptide.

These and other objects of the present application will become morereadily apparent from the detailed description given hereinafter.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The peptides of the present invention are not necessarily required tohave exactly the same amino acid sequence as represented by any one ofSEQ ID NO.1 to SEQ ID NO.8, provided that they have the osteogeneticactivity. In other words, so long as the peptides have the osteogeneticactivity, one to several amino acids in the sequences may optionally bedeleted or substituted or one to several amino acids may optionally beadded to the sequences, by a usual technique in genetic engineering orin peptide synthesis. The optionally deleted, substituted or added aminoacids may be selected as appropriate depending on the kind of aminoacids, a site and the like.

In the present invention, to “have the osteogenetic activity” may beconstrued as activity of accelerating the activation of alkalinephosphatase in osteoblasts (Yamaguchi,A., Molecular Medicine, Vol.30,No.10, 1232(1993)) so as to form neogenetic bone or induce growth ofexisting bone.

In the present specification, amino acid residues are represented byabbreviatory symbols as follows:

Ala: L-alanine residue

Asn: L-asparagine residue

Cys: L-cysteine residue

Gin: L-glutamine residue

Glu: L-glutamic acid residue

Ile: L-isoleucine residue

Leu: L-leucine residue

Lys: L-lysine residue

Pro: L-proline residue

Ser: L-serine residue

Thr: L-threonine residue

Val: L-valine residue

Glx: L-L-glutamine residue or L-glutamic acid residue

Xaa: amino acid defined in each sequence

Also in the present specification, the amino acid sequence of a peptideis written according to the conventional notation, with an amino groupat the N-terminal appearing on the left hand of the sequence andcarboxyl group at the C-terminal appearing on the right hand thereof.

The amino acid sequences represented by SEQ ID NO.1 to SEQ ID NO.8 mayalso be represented by the formula:

—Y1-Asn-Y2-Y3-Y4-Pro-Lys-Y5-Cys-Cys-Y6-Pro-Thr-Y7-Le u-Y8-Ala-Y9—,

wherein Y1 is a peptide residue or an amino acid residue selected fromthe group consisting of Asn-Ser-Val and Ile, Y2 is an amino acid residueor a peptide residue selected from the group consisting of Ser andPro-Glu, Y3 is an amino acid residue selected from the group consistingof Lys, Ser and Thr, Y4 is an amino acid residue selected from the groupconsisting of Ile and Val, Y5 is an amino acid residue selected from thegroup consisting of Ala and Pro, Y6 is an amino acid residue selectedfrom the group consisting of Ala and Val, Y7 is an amino acid residueselected from the group consisting of Glu and Gln, Y8 is an amino acidresidue selected from the group consisting of Ser and Asn, Y9 is anamino acid residue or a peptide residue selected from the groupconsisting of Ile and Ile-Ser.

The peptides of the present invention may be produced by a methodusually used for synthesizing peptides, for example, by a solid phasesynthesis method or by a liquid phase synthesis method. The solid phasesynthesis method is simpler in operation (for example, see “Sequel toBiochemical Experiments 2, Chemistry about Protein (the second volume)”p.p.641-694 edited by the Biochemical Society in Japan published on May20, 1987 by Tokyo Kagaku Dojin, Japan and “Solid Phase PeptideSynthesis—A Practical Method” p.p.152-154by Atherton, E. et al.published in 1989 by IRL Press, Oxford). The solid phase synthesis canbe carried out usually by protecting amino groups with appropriateprotecting groups, for example, either Boc (tert-butoxycarbonyl) or Fmoc(9-fluorenylmethyloxycarbonyl), or a combination thereof.

For producing the peptide of the present invention, for example, 1) anamino acid corresponding to the C-terminal of the peptide to be producedis bonded to a solid phase material insoluble to a reaction solvent viaan α-COOH group of the amino acid; 2) subsequently, in the direction tothe N-terminal of the peptide, a corresponding amino acid or peptidefragment is bonded by condensation to the amino acid of 1) afterprotecting other functional groups such as an α-amino group of thecorresponding amino acid or peptide fragment other than an α-COOH group;3) a protecting group of an amino group forming a peptide bond such asan α-amino group is removed from the bonded amino acid or peptidefragment; these steps are repeated to elongate a peptide chain in orderto form a peptide chain corresponding to the desired peptide.

The thus produced peptide chain is detached from the solid phasematerial and protecting groups are removed from protected functionalgroups. Subsequently the peptide chain is purified, thereby to obtainthe desired peptide.

Here, as the solid phase material, styrene-divinyl benzene copolymers,Merrifield resins, chloromethyl resins, Wang resins, Sieber resins, rinkamide resins, rink acid resins, 2-chlorotrityl chloride resins,HMBA-MBHA resins, MBHA resins, oxime resins and the like may be used.Among these resins, styrene-divinyl benzene copolymers are preferred.

It is preferred from the viewpoint of preventing side reaction that thedetachment of the peptide chain from the solid phase material and theremoval of the protecting groups are carried out simultaneously usingtrifluoroacetic acid or hydrogen fluoride.

As a solvent and a condensing agent in the peptide synthesis, any onesusually known in the art may be used as required. For example, DMF(dimethylformamide), trichloroethanol, N-methylpyrrolidone and the likemay be mentioned as solvents, and DCC, HATU(0-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate), HOBt (1-hydroxybenzotriazole), HBTU(0-benzotriazole-1-yl-N,N,N′,N′-tetramethyl uroniumhexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluorophosphate), CF₃-NO₂-PyBOP and the like may be mentioned ascondensing agents.

For purifying the obtained peptide, it is effective to utilize reversephase liquid chromatography.

Either or both of the N- and C-terminals of the peptide of the presentinvention may optionally be modified chemically. For example, theN-terminal may be acetylated and the C-terminal may be amidated.

The peptide of the present invention may form a physiologicallyacceptable salt by conventional salt formation reaction. Such salts caninclude salts with inorganic acids such as hydrochloric acid, sulfuricacid and phosphoric acid; salts with organic acids such as lactic acid,tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid,citric acid, oleic acid and palmitic acid; salts with hydroxides andcarbonates of alkali metals and alkali earth metals such as sodium,potassium, calcium and aluminum; and salts with amines such astriethylamine, benzylamine, diethanolamine, t-butylamine,dicyclohexylamine and arginine.

Preferred examples of peptides provided by the present invention arepeptides having amino acid sequences represented by SEQ ID NO.9 or SEQID NO.10, which are examples of SEQ ID NO.1 and peptides having an aminoacid sequence represented by SEQ ID NO. 11, which is an example of SEQID NO.8, and particularly peptides having amino acid sequencesrepresented by SEQ ID NO. 9 to SEQ ID NO.11 and having an amino group atthe N-terminal and a carboxyl group at the C-terminal, and peptideshaving an amino acid sequence represented by SEQ ID NO.9 and having anacetyl group at the N-terminal and a carboxyl group at the C-terminal.

These peptides may have a part of thereof deleted or substituted or haveaddition to amino acid(s) thereto so long as they have the osteogeneticactivity, as described above.

It is verified that the peptide of the present invention has theosteogenetic activity and is negligible in toxicity such as cytotoxity,systemic acute toxicity and the like. As for the osteogeneticaccelerator comprised of the peptide of the present invention, it ispossible to avoid the problems of infection and antigenicity resultingfrom matrices by means of sterilizing operation such as γ-raysterilization, moist heat vapor sterilization and selection of a carriermade of a polysaccharide having low antigenicity or the like duringproduction process.

The peptide of the present invention may be used singly for the purposeof preventing or treating bone fractures. Also the peptide may be usedin the form of an osteogenetic accelerator obtained by fixing, mixing,solving or suspending the peptide in a proper carrier or an aqueoussolvent which can contain a variety of pharmacologically acceptableadditives such as a stabilizer, a preservative, a thickener, asolubilizer and the like. It is particularly preferable that theosteogenetic accelerator of the present invention is one in which thepeptide is fixed to a carrier.

The carrier for fixing the peptide of the present invention is notparticularly limited to any type provided that it has compatibility toliving bodies. For example, it is possible to use, singly or incombination, carriers which can be degraded and absorbed in vivo, suchas covalently crosslinked gels of alginate (Suzuki,Y. et al., J. Biomed.Mater. Res., 39, 317(1998)), gels of protein such as collagen,hyaluronic acid, calcium sulfate, polylactic acid, polyglycolic acid,hydroxyapatite, tricalcium phosphate and the like, as well as variousceramics and artificial bone. In addition, starch gel, chitin/chitosangel, agarose gel and dextran gel may be used as polysaccharide gels.Among these carriers, a covalently crosslinked gel of alginate and a gelof hyaluronic acid are preferred from the view point of non-inflammatory(J.Biomed.Mater.Res.Appl.Biomater., 48, 522-527 (1999) andJ.Artif.Organs, 1, 28-32 (1998)) and non-immunogenic(J.Biomed.Master.Res., 1994, Sep;28(9):1037-46) properties.

The method of fixing the peptide on a carrier is not particularlylimited. It is possible to adopt a fixation method allowing formation ofcovalent bond, ionic bond, hydrophobic bond, hydrogen bond, SS bond orthe like, for example, an immersion, impregnation, spray, applicationand dropping method with use of a solution containing the peptide. Amongthese fixation methods, fixation by covalent bond is preferred from theviewpoint of stability and continuity of effect. Such fixation can bedone by a method usually used for fixing a physiologically activeprotein such as an enzyme (for example, see Scouten, W. H., Methods inEnzymol., 135, Mosbach, K. Ed., 1987, Academic Press NY, p.p.30-65).

Preferably, the peptide to be fixed is used in an amount of about 0.01to about 50 parts by weight, preferably about 0.1 to 25 parts by weight,with respect to 100 parts by weight of a dry carrier. The peptide thusfixed is usually used for treatment of a fracture or the like by beingimplanted in a deficient site in bone. If the peptide is used in anamount smaller than 0.01 parts by weight with respect to 100 parts byweight of a dry carrier, the effect of the peptide tends to beinsufficient. If the peptide is used in an amount larger than 50 partsby weight, on the other hand, the ratio of fixation of the peptide tothe carrier declines and the peptide tends not to be utilizedeffectively.

As aqueous solvents, physiological saline and physiologically acceptableaqueous solutions of mannitol, sucrose, lactose, maltose, glucose,fructose or the like. A glucose aqueous solution of 5% and aphysiological saline are preferable. These aqueous solvents may be usedso that the concentration of the peptide is 0.001% to 5%, preferably0.01% to 1%. If the concentration of the peptide exceeds 5%, theviscosity of the solution rises. Accordingly, there are tendencies thatadministration becomes difficult and that the peptide separates at anadministration site and as a result its effect declines. If theconcentration of the peptide is below 0.001%, on the other hand, theeffect of the peptide tends to be insufficient.

The osteogenetic accelerator may be used by intravenous, subcutaneous,intraperitoneal, intra-articular or dermal administration or by fillingit in a defective site in bone. Further, if capsulated or made intoliposomes by the conventional method, the osteogenetic accelerator canbe administered orally.

Thus, the peptide and the osteogenetic accelerator of the invention canpromote treatment of fractures by being administered to patients withfractures caused by rheumatoid arthritis and osteoporosis or by beingfilled or implanted in a defective site in bone. Also they can inhibitdecrease in bone substance and prevent fractures by being administeredto patients with rheumatoid arthritis, osteoporosis and periodonticdiseases.

The dose of the peptide as an active ingredient may vary as requireddepending upon the weight of bone desired to be formed, the site ofinjured bone, the condition of bone, and the age, sex and weight of apatient and the like. But usually, the peptide expresses its effect bybeing administered at a dose of 0.01 μg/kg to 33 mg/kg (for an adult),preferably 0.01 μg/kg to 3.3 mg/kg (for an adult), once per day.

EXAMPLES

The present invention is now described by way of examples, which shouldnot be construed to limit the scope of the invention.

Example 1

A peptide having the amino acid sequence of SEQ ID NO. 9 which had anamino group at the N-terminal and a carboxyl group at the C-terminal wassynthesized by the solid phase synthesis method using an automaticpeptide synthesizer.

More particularly, with use of 0.1 mmol of particulate resin (producedby US Applied Biosystems, HMP isoleucine) comprised ofstyrene-divinylbenzene copolymer (the molar composition ratio of styreneto divinylbenzene was 99:1) containing4-(N^(α)-9-(fluorenylmethoxycarbonyl)-L-isoleucyl)-oxymethyl-phenoxy-methylgroup in a proportion of 0.65 mmol/g-resin, successively bonded wereamino acids corresponding in the direction from the carboxyl terminal tothe amino terminal of the peptide.

In bonding reaction, used as amino acids wereN^(α)-9-(fluorenylmethoxycarbonyl)-N^(β)-trityl-L-asparagine (Fmocasparagine), N^(α)-9-(fluorenylmethoxycarbonyl)-0-t-butyl-L-serine (Fmocserine), N^(α)-9-(fluorenylmethoxycarbonyl)-valine (Fmoc-valine),N^(α)-9-(fluorenylmethoxycarbonyl)-N^(ε)-t-butyloxycarbonyl-L-lysine(Fmoc lysine), N^(α)-(fluorenylmethoxycarbonyl)-L-isoleucine (Fmocisoleucine), N^(α)-(fluorenylmethoxycarbonyl)-L-proline (Fmoc proline),N^(α)-(fluorenylmethoxycarbonyl)-L-alanine (Fmoc Alanine),N^(α)-9-(fluorenylmethoxycarbonyl)-S-trityl-L-cysteine (Fmoc cysteine),N^(α)-9-(fluorenylmethoxycarbonyl)-0-t-butyl-L-threonine (Fmocthreonine), N^(α)-9-(fluorenylmethoxycarbonyl)-^(γ)-butyl-L-glutamicacid (Fmoc glutamic acid), andN^(α)-(fluorenylmethoxycarbonyl)-L-leucine (Fmoc leucine), all beingproduced by US applied Biosystems, in the amount of 1 mmol in eachbonding step.

As the condensing agent and additive for peptide synthesis, HBTU andHOBt were used.

The resulting peptide resin was treated with 10 ml of trifluoroaceticacid containing 2.5% of water and 2.5% of ethanedithiol for three hours.The resulting solution was added to diethyl ether. The generatedprecipitate was further washed with diethyl ether several times in orderto deprotect the peptide and detach it from the resin. The resultingcrude product was purified by preparative RP-HPLC (column: Novapak HRC18 25×100 mm, RCM 25×10 with a pressure module produced by NipponWaters Kabushiki Kaisha, Japan) using a mixture solvent of water andacetonitrile (with varying the concentration of acetonitrile from 5 vol% to 50 vol % in 30 minutes) containing trifluoroacetic acid in theproportion of 0.05%.

The resulting purified peptide was subjected to an AKTA explorer 1OXTproduced by Pharmacia Biotech Kabushiki Kaisha, Japan (column : μRPCC2/C18 ST4.6/100 produced by Pharmacia Biotech Kabushiki Kaisha, mobilephase: a mixture solvent of water and acetonitrile containing 0.05 vol %of trifluoroacetic acid (with varying the concentration of acetonitrilefrom 5 vol % to 50 vol % in 30 minutes), a flow rate: 0.5 ml/min.). Asingle peak was observed at 24.5 min.

The molecular weight of the purified peptide was found to be 2,075 byFAB mass spectrometry (theoretical molecular weight : 2,074.45).Therefore, it was verified that the desired peptide was obtained.

Examples 2 to 4

A peptide (Example 2) having the amino acid sequence of SEQ ID NO. 9 andhaving an acetyl group at the N-terminal and a carboxyl group at theC-terminal, a peptide (Example 3) having the sequence of SEQ ID NO. 10and having an amino group at the N-terminal and a carboxyl group at theC-terminal, and a peptide (Example 4) having the sequence of SEQ ID NO.11 which had an amino group at the N-terminal and a carboxyl group atthe C-terminal were synthesized in the same manner as in Example 1.

In Example 2, a peptide resin obtained by the automatic peptidesynthesizer was suspended in 20 ml of dimethylformamide with stirring,to which 2 ml of acetic anhydride and 26 μl of diisopropylethylaminewere added, followed by stirring at room temperature for another fourhours. The acetylated peptide resin was well washed on a glass filterwith methanol, and then dried under reduced pressure.

In Example 4, used was 0.1 mmol of particulate resin (produced by USApplied Biosystems, HMP serine) comprised of styrene-divinylbenzenecopolymer (the molar composition ratio of styrene to divinylbenzene was99 1) containing4-(N^(α)-9-(fluorenylmethoxycarbonyl)-0-t-butyl-L-serin)-oxymethyl-phenoxy-methyl group in a proportion of 0.65 mmol/g-resin.

As an amino acid, used wasN^(α)-9-(fluorenylmethoxycarbonyl)-N^(γ)-trityl-L-glutamine (Fmocglutamine) produced by US Applied Biosystems in addition to the aminoacids used in Example 1.

The resulting peptide resins were subjected to deprotection anddetachment from the solid phase and the resulting crude products werepurified, in the same manner as in Example 1. The purified peptides wereeach examined on elution time and molecular weight by analytical HPLCand by FAB mass spectroscopy. The results are shown in Table 1.

TABLE 1 Theoretical Elution Molecular Molecular Examples Time WeightWeight Example 2 25.2 min 2117 2116.49 Example 3 25.0 min 2033 2033.36Example 4 22.1 min 2097 2096.46

Test Example 1 Determination of Alkali Phosphatase Activity in BoneMarrow Cells of Rats—in Vitro

Femurs were aseptically taken out of six-week-old female rats (Lewis,purchased from Nippon SLC). Both ends of the femurs were cut off. Thenbone marrow was extruded into centrifugal tubes by means of Eagle's MEMcontaining 10% of fetal bovine serum using an injection syringe with a21G injection needle. The bone marrow cells were sufficiently dispersedwith a pipette and filtered by a 40 μm filter to remove agglutinations.The resulting bone marrow cells were centrifuged with Eagle's MEMcontaining 10% of fetal bovine serum three times (1200 rpm, 5 min.×3),and then the number of cells was counted. The bone marrow cells werediluted with Alpha MEM containing 10% of fetal bovine serum so thatrelatively large bone marrow cells were present in a concentration of10⁶/ml. The dilution was pipetted 10 ml in each culture flask having aculture area of 25 cm² and incubated at 37° C. in the presence of 5%CO₂.

Twenty-four hours after the incubation was started, medium was replacedwith Alpha MEM containing 10% of fetal bovine serum and the peptideobtained in Example 1. Thereafter, the medium was replaced similarlyevery three days.

One week after the incubation was started, multiplied cells were stainedwith an alkaline phosphatase staining kit (produced by Sigma).

The results showed remarkable increase in the alkaline phosphataseactivity as compared with cases where human IL-1 was added in aconcentration of 10 ng/ml instead of the peptide obtained in Example 1and with cases where nothing was added instead of the peptide obtainedin Example 1.

The same test was carried out on the peptides obtained in Examples 2 to4, and as a result, remarkable increase in the alkaline phosphataseactivity was observed as in the case of the peptide of Example 1.

Test Example 2 Determination of Alkaline Phosphatase Activity in BoneMarrow Cells of Rats—in Vivo

A solution of 500 μg of the peptide obtained in Example 1 in phosphatebuffer (PBS: 10 mM, containing 0.15M of sodium chloride, pH: 7.4) wasintraperitoneally administered to six-week-old female rats (purchasedfrom Nippon SLC, Japan) three times in a week. After one week, femurs ofthe rats were taken out aseptically. Both ends of the femurs were cutoff. Then bone marrow was extruded into centrifugal tubes by means ofEagle—MEM containing 10% of fetal bovine serum using an injectionsyringe with a 21G injection needle. The bone marrow cells weresufficiently dispersed with a pipette and filtered by a 40 μm filter toremove agglutinations. The resulting bone marrow cells were centrifugedwith Eagle's MEM containing 10% of fetal bovine serum three times (1200rpm, 5 min.×3), and then the number of cells was counted. The bonemarrow was diluted with Alpha MEM containing 10% of fetal bovine serumso that relatively large bone marrow cells were present in aconcentration of 10⁶/ml. The dilution was pipetted on a 24-hole cultureplate, 1 ml per well, and incubated at 37° C. in the presence of 5% CO₂.

Twenty-four hours after the incubation was started, medium was replacedwith Alpha MEM containing 10% of fetal bovine serum. Thereafter, themedium was replaced similarly every three days.

One week after the incubation was started, the culture plate was clearedof supernatant and washed with PBS once. In each well of the 24-wellplate, 1 ml of Tris buffer (20 mM, pH 8.5) containing 1% Triton X-100was added and allowed to stand for 30 minutes to dissolve cells.

With respect to 100 μl of the resulting cell solution, 100 μl of Trisbuffer (1.5 M, containing 1 mM of ZnCl₂ and 1 mM of MgCl₂, pH 8.5)containing 7.5 mM of p-nitrophenyl phosphate were added. Increase inabsorbency at405 nm was measured to determine the alkaline phosphataseactivity in the cell solution. The concentration of protein in the cellsolution was also determined using a BCA assay kit (produced by Pierce).

As a result, the alkaline phosphatase activity in the rats having beenadministered the peptide of Example 1 was 6.3±1.4 μmol/min.mg-protein,while that in rats having received administration only of PBS instead ofthe peptide of Example 1 was 3.2±1.1 μmol/min.mg-protein. It was foundthat the peptide of Example 1 brought about remarkable increase in thealkaline phosphatase activity.

Example 5

Ethylene diamine (EDA, produced by Wako Jun'yaku Kogyo Kabushiki Kaisha,Japan), 0.6 g (10mmol), dissolved in 10 ml of methanol was dropped into150 ml of methanol in which 2.3 g (20 mmol) of N-hydroxysuccinimide(HOSu, produced by KK Peptide Kenkyusho, Japan) had been dissolved,while stirring at room temperature. After dropping, the mixture wasstirred for another one hour. Precipitated crystals were taken byfiltration and dried under reduced pressure, to obtain 2.6 g (a yield ofabout 90%) of ethylenediamine 2N-hydroxysuccinimide salt (EDA.2HOSu).

EDA. 2HOSu, 66 mg, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (WSCD.HCl, produced by KK Peptide Kenkyusho), 0.48g, weredissolved in 30 ml of 1 wt % aqueous solution of sodium alginate(produced by Funakoshi Kabushiki Kaisha, viscosity: 550 cp, M/G ratio:1.0). The resulting mixture was cast on a 10 cm×10cm Teflon-coatedaluminum tray and allowed to stand at 25° C. for 48 hours, to obtain acovalently crosslinked gel of alginate.

The gel was sufficiently washed with a purified water for injection(produced by Otsuka Seiyaku) in which 2.5 mM of CaCdl₂ and 143 mM ofNaCl had been dissolved and then washed only with the purified water forinjection. The alginate gel after washing was freeze-dried to obtain awhite sponge-like gel.

The resulting sponge-like gel, 0.1 g, was immersed in 20 ml ofdimethylformamide, to which 12 mg of N-hydroxysuccinimide and 19 mg ofWSCD.HCl were added, and shook at room temperature overnight. Thesponge-like gel was well washed with methanol and dimethylformamide, towhich 10 ml of dimethylformamide solution containing 20 mg of thepeptide obtained in Example 1 was added, and shook at room temperatureovernight. The resulting gel was well washed with methanol and ethanol,to obtain an osteogenetic accelerator in which the peptide of Example 1was fixed.

Test Example 3 Intramuscular Implant Test on Rats

The osteogenetic accelerator obtained in Example 5, 0.01 g, wasimplanted in femoral muscle of six-week-old female SD rats (purchasedfrom Nippon SLC). After three weeks, peripheral tissue includingimplants was taken out and subjected to tissue staining (see “ClinicalTest Techniques” edited by Kanno,T., Matsuda,N., {circle around (5)}Pathology.Pathological Tissue Cytology, published by Igakushoin, Japanin 1991).

As a result, von Kossa staining revealed that calcium deposited onimplant sites. For comparison, a sponge-like gel not having the peptidefixed thereon was implanted on the opposite side of the identical rats,where deposition of calcium was not recognized at all through the vonKossa staining.

Test Example 4 Deficient Bone Site Implant Test on Dogs

The osteogenetic accelerator obtained in Example 5, 0.07 g, wasimplanted in deficient sites of 7 mm diameter which had beenartificially formed in mandibles of six-month-old female beagles(purchased from Nippon SLC). Two weeks after implantation, tissueincluding the implant sites was taken out and subjected to tissuestaining. Formation of neogenetic bone was obviously observed. Forcomparison, a sponge-like gel not having the peptide fixed thereon wasimplanted on the opposite side of the identical dogs, where anyneogenetic bone was not recognized at all.

Test Example 5 Cytotoxicity Test in Vitro—Rat Bone Marrow Cells

Femurs were aseptically taken out of six-week-old female rats (Lewis,purchased from Nippon SLC). Both ends of the femurs were cut off. Thenbone marrow was extruded into centrifugal tubes by means of Eagle's MEMcontaining 10% of fetal bovine serum using an injection syringe with a21G injection needle. The bone marrow cells were sufficiently dispersedwith a pipette and filtered through a 40 μm filter to removeagglutinations. The resulting bone marrow cells were centrifuged withEagle's MEM containing 10% of fetal bovine serum three times (1200 rpm,5 min.×3), and then the number of cells was counted. The bone marrowcells were diluted with Alpha MEM containing 10% of fetal bovine serumso that relatively large bone marrow cells were present in aconcentration of 10⁶/ml. The dilution was pipetted 10 ml in each cultureflask having a culture area of 25 cm² and incubated at 37° C. in thepresence of 5% CO₂. To the resulting dilution, a solution of the peptidedescribed in Example 1 in phosphate buffer (PBS: 10 mM, containing 0.15Mof sodium chloride, pH: 7.4) was added so that the resultingconcentration became 30 μg/ml.

Twenty-four hours after the incubation was started, culture medium wasremoved and Alpha MEM containing 10% of fetal bovine serum and 30 μg/mlof the peptide described in Example 1 was added to the cells underculture. Thereafter, the medium was replaced similarly every two days.Incubation was continued for seven days. After seven days, the number offormed colonies of bone marrow cells was counted using Giemsa staining.The number of colonies was 101 in average with flasks to which thepeptide of Example 1 had been added, and it was 89 in average withflasks to which PBS has been added instead of the peptide of Example 1in the same amount. The results showed that the peptide of Example 1 didnot prevent growth of bone marrow cells and that it did not havecytotoxicity.

Test Example 6 Cytotoxicity Test in Vitro—C3H10T1/2 Cells

C3H10T1/2 cells were dispersed in Eagle's MEM containing 10% fetalbovine serum, pipetted on a 96-well culture plate, 3750 cells per well,and incubated at 37° C. in the presence of 5% CO₂. Into the wells, a PBSsolution of the peptide described in Example 1 was added so that theresulting concentration became 100 μg/ml, followed by incubation underthe same conditions for six days. Then the culture medium was removedand a cytolytic solution (0.1M Tris-HCl buffer containing 1% TritonX-100, pH9) was added to dissolve cells. Then the concentration ofprotein was determined using BCA reagent (produced by FunakoshiKabushiki Kaisha).

The concentration of protein was 0.12±0.02 mg/ml (n=12) with wells towhich the peptide of Example 1 had been added, and it was 0.07±0.03mg/ml (n=12) with wells to which PBS has been added instead of thepeptide of Example 1 in the same amount. The results showed that thepeptide of Example 1 did not prevent growth of C3H10T1/2 cells and thatit did not have cytotoxicity.

Test Example 7 Acute Toxicity Test on Rats

A PBS solution of 500 μg of the peptide described in Example 1 wasintraperitoneally administered to six-week-old female rats (Lewis,purchased from Nippon SLC) three times in a week. After a week, a grouphaving received administration of the peptide of Example 1 showed anincrease in weight of 25±6 g (n=6), and a group having receivedadministration of the same amount of PBS instead of the peptide ofExample 1 showed an increase in weight of 23±5 g (n=6). It was notrecognized that the administration of the peptide of Example 1 preventedincrease in weight. Neither were the rats observed to have a poor coatof fur or any abnormal behavior during the test. Therefore, the peptideof Example 1 showed no significant acute toxicity.

According to the present invention, provided are a peptide having theosteogenetic activity in which various side-effects such as cytotoxityare alleviated and an osteogenetic accelerator containing the peptide asan active ingredient. The peptide and the osteogenetic accelerator areuseful for treating fractures, filling defective sites in bone,controlling reduction in bone substance involved with osteoporosis andperiodontal diseases and preventing fractions associated withosteoporosis and rheumatoid arthritis.

Free Text for Sequence Listing

Each “Xaa” in SEQ ID NO. 1 to SEQ ID No. 8 in the SEQUENCE LISTINGindicates amino acids defined as defined below:

In SEQ ID NO. 1 and SEQ ID NO. 2, Xaa at position 6 represents Lys, Seror Thr, Xaa at position 7 represents Ile or Val, Xaa at position 10represents Ala or Pro, Xaa at position 13 represents Ala or Val, and Xaaat position 18 represents Ser or Asn.

In SEQ ID NO. 3 and SEQ ID NO. 4, Xaa at position 7 represents Lys, Seror Thr, Xaa at position 8 represents Ile or Val, Xaa at position 11represents Ala or Pro, Xaa at position 14 represents Ala or Val, and Xaaat position 19 represents Ser or Asn.

In SEQ ID NO. 5 and SEQ ID NO. 6, Xaa at position 4 represents Lys, Seror Thr, Xaa at position 5 represents Ile or Val, Xaa at position 8represents Ala or Pro, Xaa at position 11 represents Ala or Val, and Xaaat position 16 represents Ser or Asn.

In SEQ ID NO. 7 and SEQ ID NO. 8, Xaa at position 5 represents Lys, Seror Thr, Xaa at position 6 represents Ile or Val, Xaa at position 9represents Ala or Pro, Xaa at position 12 represents Ala or Val, and Xaaat position 17 represents Ser or Asn.

11 1 20 PRT Artificial Sequence Synthesized peptide 1 Asn Ser Val AsnSer Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr Glx 1 5 10 15 Leu Xaa AlaIle 20 2 21 PRT Artificial Sequence Synthesized peptide 2 Asn Ser ValAsn Ser Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr Glx 1 5 10 15 Leu XaaAla Ile Ser 20 3 21 PRT Artificial Sequence Synthesized peptide 3 AsnSer Val Asn Pro Glu Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr 1 5 10 15Glx Leu Xaa Ala Ile 20 4 22 PRT Artificial Sequence Synthesized peptide4 Asn Ser Val Asn Pro Glu Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr 1 5 1015 Glx Leu Xaa Ala Ile Ser 20 5 18 PRT Artificial Sequence Synthesizedpeptide 5 Ile Asn Ser Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr Glx LeuXaa 1 5 10 15 Ala Ile 6 19 PRT Artificial Sequence Synthesized peptide 6Ile Asn Ser Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr Glx Leu Xaa 1 5 1015 Ala Ile Ser 7 19 PRT Artificial Sequence Synthesized peptide 7 IleAsn Pro Glu Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr Glx Leu 1 5 10 15Xaa Ala Ile 8 20 PRT Artificial Sequence Synthesized peptide 8 Ile AsnPro Glu Xaa Xaa Pro Lys Xaa Cys Cys Xaa Pro Thr Glx Leu 1 5 10 15 XaaAla Ile Ser 20 9 20 PRT Artificial Sequence Synthesized peptide 9 AsnSer Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu 1 5 10 15Leu Ser Ala Ile 20 10 20 PRT Artificial Sequence Synthesized peptide 10Asn Ser Val Asn Ser Ser Ile Pro Lys Ala Cys Cys Val Pro Thr Glu 1 5 1015 Leu Ser Ala Ile 20 11 20 PRT Artificial Sequence 11 Ile Asn Pro GluThr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu 1 5 10 15 Asn Ala IleSer 20

What is claimed is:
 1. A synthesized peptide consisting of any one ofthe sequences SEQ ID NO:
 1. 2. The synthesized peptide according toclaim 1 consisting of any one of the sequences SEQ ID NO:9 and SEQ IDNO:
 10. 3. An osteogenetic accelerator comprising the peptide set forthin claim 1, or a pharmalogically acceptable salt thereof, attached to abiocompatible carrier.
 4. The osteogenetic accelerator according toclaim 3, wherein the carrier is selected from a group consisting of aceramic, an artificial bone, a covalently cross-linked gel of alginate,and a gel of collagen, hyaluronic acid, calcium sulfate, polylacticacid, polyglycolic acid, hydroxyapatite, tricalcium phosphate, starch,chitin/chitosan, agarose, or dextran.
 5. The osteogenetic acceleratoraccording to claim 3 which contains 0.01 to 50 parts by weight of thepeptide per 100 parts by weight of the carrier.
 6. An osteogeneticaccelerator comprising the peptide of claim 1, or a pharmacologicallyacceptable salt thereof, mixed with, dissolved in, or suspended inaqueous solvent.
 7. The osteogenetic accelerator according to claim 6,wherein the aqueous solvent is physiological saline solution or aphysiologically acceptable aqueous solution selected from a groupconsisting of mannitol, sucrose, lactose, maltose, glucose, andfructose.
 8. The osteogenetic accelerator according to claim 6 or 7,wherein the concentration of the peptide is 0.001% to 5% with respect tothe aqueous solvent.
 9. The osteogenetic accelerator as set forth inclaim 3 which is used for treating a bone fracture by inducing boneformation at the fracture site or for inhibiting a decrease in bonesubstance.
 10. The peptide of claim 1, wherein the peptide N-terminal isacetylated, or the peptide C-terminal is amidated, or both theN-terminal is acetylated and the C-terminal is amidated.
 11. Asynthesized peptide consisting of the sequence SEQ ID NO:9.
 12. Anosteogenetic accelerator comprising the peptide set forth in claim 11,or a pharmacologically acceptable salt thereof, attached to abiocompatible carrier.
 13. The osteogenetic accelerator according toclaim 12, wherein the carrier is selected from a group consisting of aceramic, an artificial bone, a covalently cross-linked gel of alginate,and a gel of collagen, hyaluronic acid, calcium sulfate, polylacticacid, polyglycolic acid, hydroxyapatite, tricalcium phosphate, starch,chitin/chitosan, agarose, or dextran.
 14. The osteogenetic acceleratoraccording to claim 12 which contains 0.01 to 50 parts by weight of thepeptide per 100 parts by weight of the carrier.
 15. An osteogeneticaccelerator comprising the peptide of claim 11, or a pharmacologicallyacceptable salt thereof, mixed with, dissolved in, or suspended inaqueous solvent.
 16. The osteogenetic accelerator according to claim 15,wherein the aqueous solvent is physiological saline solution or aphysiologically acceptable aqueous solution selected from a groupconsisting of mannitol, sucrose, lactose, maltose, glucose, andfructose.
 17. The osteogenetic accelerator according to claim 15 or 16,wherein the concentration of the peptide is 0.001% to 5% with respect tothe aqueous solvent.
 18. The osteogenetic accelerator as set forth inclaim 12 which is used for treating a bone fracture by inducing boneformation at the fracture site or for inhibiting a decrease in bonesubstance.
 19. The peptide of claim 11, wherein the peptide N-terminalis acetylated, or the peptide C-terminal is amidated, or both theN-terminal is acetylated and the C-terminal is amidated.
 20. Anosteogenetic accelerator comprising a physiologically acceptable salt ofthe peptide set forth in claim
 11. 21. A synthesized peptide consistingof the sequence SEQ ID NO:10.
 22. An osteogenetic accelerator comprisingthe peptide set forth in claim 21, or a pharmacologically acceptablesalt thereof, attached to a biocompatible carrier.
 23. The osteogeneticaccelerator according to claim 22, wherein the carrier is selected froma group consisting of a ceramic, an artificial bone, a covalentlycross-linked gel of alginate, and a gel of collagen, hyaluronic acid,calcium sulfate, polylactic acid, polyglycolic acid, hydroxyapatite,tricalcium phosphate, starch, chitin/chitosan, agarose, or dextran. 24.The osteogenetic accelerator according to claim 22 which contains 0.01to 50 parts by weight of the peptide per 100 parts by weight of thecarrier.
 25. An osteogenetic accelerator comprising the peptide of claim21, or a pharmacologically acceptable salt thereof, mixed with,dissolved in, or suspended in aqueous solvent.
 26. The osteogeneticaccelerator according to claim 25, wherein the aqueous solvent isphysiological saline solution or a physiologically acceptable aqueoussolution selected from a group consisting of mannitol, sucrose, lactose,maltose, glucose, and fructose.
 27. The osteogenetic acceleratoraccording to claim 25 or 26, wherein the concentration of the peptide is0.001 % to 5% with respect to the aqueous solvent.
 28. The osteogeneticaccelerator as set forth in claim 22 which is used for treating a bonefracture by inducing bone formation at the fracture site or forinhibiting a decrease in bone substance.
 29. The peptide of claim 21,wherein the peptide N-terminal is acetylated, or the peptide C-terminalis amidated, or both the N-terminal is acetylated and the C-terminal isamidated.
 30. An osteogenetic accelerator comprising a physiologicallyacceptable salt of the peptide set forth in claim 21.